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Combined use of vitamin d derivatives and anti-proliferative agents for treating bladder cancer

Combined use of vitamin d derivatives and anti-proliferative agents for treating bladder cancer description/claims

Bladder cancer comprises several disease subtypes: transitional cell carcinoma CCC), squamous cell carcinoma (SCC), adenocarcinoma. Of these, TCC is predominant, accounting for 90% of all bladder carcinomas. Both TCC and SCC can be of a non-invasive or invasive type, and they are collectively known as superficial bladder cancer (see Schenkman & Lamm Scientific World Journal. 2004 (28):4 Suppl 1:387-99). Such cancers can be surgically removed (typically by transurethral resection), but have a great tendency for recurrence. The incidence of bladder cancer increases with age. People over the age of 70 develop the disease 2 to 3 times more often than those aged 55-69, and 15 to 20 times more often than those aged 30-54. Bladder cancer is 2 to 3 times more common in men compared to women. In the United States, approximately 38,000 men and 15,000 women are diagnosed with the disease each year. Bladder cancer is the fourth most common type of cancer in men and the eighth most common type in women.

As mentioned above, superficial bladder cancer can be surgically removed, but has a great tendency for recurrence (see Schenkman & Lamm Scientific World Journal. 2004 (28); 4 Suppl 1:387-99).

Anthracyclines such as doxorubicin and epirubicin are known for the treatment of bladder cancer. Administration of anthracyclines is believed to reduce the risk of recurrence following surgery however these compound have considerable systemic toxicity. Toxicity can be reduced by instilling the drug directly into the bladder, however it can still enter the system if the bladder has in any way been damaged by the surgery. Further these compounds are believed to be potentially carcinogenic irrespective of the route of administration. Thus, there is an unmet medical need for a safer treatment which prevents recurrence or progression to invasive disease.

As described herein, it has now surprisingly been found that vitamin D compounds when used in combination with one or more other anti-proliferative agents, for example an anthracycline compound such as doxorubicin or epirubicin, inhibit proliferation of bladder cancer cells and may therefore be expected to treat and prevent bladder cancer, in particular superficial bladder cancer.

Calcitriol (1,25 dihydroxycholecalciferol; the active, hormonal form of vitamin D) and its analogues can have significant anti-proliferative effects on various tumor cell lines, but clinical use as an anti-cancer agent can be limited by hypercalcemic liability. Superficial bladder cancer offers the unique advantage of being treatable by intravesical administration of anti-proliferative agents, thus permitting the use of high calcitriol concentrations while avoiding systemic side effects such as hypercalcemia and down-modulation of the anti-tumor immune response. Since studies in animal models have shown only marginal effects of intravesical calcitriol administration on superficial bladder cancer (see Konety et al. (2001) J. Urol. 165(1):253-258), the Inventors have examined the efficacy of combinations of calcitriol and various clinically used anti-proliferative agents.

The importance of vitamin D (cholecalciferol) in the biological systems of higher animals has been recognized since its discovery by Mellanby in 1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB) SRS 61:4). It was in the interval of 1920-1930 that vitamin D officially became classified as a “vitamin” that was essential for the normal development of the skeleton and maintenance of calcium and phosphorous homeostasis.

Studies involving the metabolism of vitamin D3 were initiated with the discovery and chemical characterization of the plasma metabolite, 25-hydroxyvitamin D3 [25(OH)D3] (Blunt, J. W. et al. (1968) Biochemistry 6:3317-3322) and the hormonally active form, 1-alpha,25(OH)2D3 (Myrtle, J. F. et al. (1970) J. Biol. Chem. 245:1190-1196; Norman, A. W. et al. (1971) Science 173:51-54; Lawson, D. E. M. et al. (1971) Nature 230:228-230; Holick, M. F. (1971) Proc. Natl. Acad. Sci. USA 68:803-804). The formulation of the concept of a vitamin D endocrine system was dependent both upon appreciation of the key role of the kidney in producing 1-alpha,25(OH)2D3 (calcitriol) in a carefully regulated fashion (Fraser, D. R. and Kodicek, E. (1970) Nature 288:764-766; Wong, R. G. et al. (1972) J. Clin. Invest. 51:1287-1291), and the discovery of a nuclear receptor for 1-alpha,25(OH)2D3 (VDR) in the intestine (Haussler, M. R. et al. (1969) Exp. Cell Res. 58:234-242; Tsai, H. C. and Norman, A. W. (1972) J. Biol. Chem. 248:5967-5975).

The operation of the vitamin D endocrine system depends on the following: first, on the presence of cytochrome P450 enzymes in the liver (Bergman, T. and Postlind, H. (1991) Biochem. J. 276:427-432; Ohyama, Y. and Okuda, K. (1991) J. Biol. Chem. 266:8690-8695) and kidney (Henry, H. L. and Norman, A. W. (1974) J. Biol. Chem. 249:7529-7535; Gray, R. W. and Ghazarian, J. G. (1989) Biochem. J. 259:561-568), and in a variety of other tissues to effect the conversion of vitamin D3 into biologically active metabolites such as I-alpha,25(OH)2D3 and 24R,25(OH)2D3; second, on the existence of the plasma vitamin D binding protein (DBP) to effect the selective transport and delivery of these hydrophobic molecules to the various tissue components of the vitamin D endocrine system (Van Baelen, H. et al. (1988) Ann. NY Acad. Sci. 538:60-68; Cooke, N. E. and Haddad, J. G. (1989) Endocr. Rev. 10:294-307; Bikle, D. D. et al. (1986) J. Clin. Endocrinol. Metab. 63:954-959); and third, upon the existence of stereoselective receptors in a wide variety of target tissues that interact with the agonist 1-alpha,25(OH)2D3 to generate the requisite specific biological responses for this secosteroid hormone (Pike, J. W. (1991) Annu. Rev. Nutr. 11: 189-216). To date, there is evidence that nuclear receptors for 1-alpha,25(OH)2D3 (VDR) exist in more than 30 tissues and cancer cell lines (Reichel, H. and Norman, A. W. (1989) Annu. Rev. Med. 40:71-78).

Vitamin D3 and its hormonally active forms are well-known regulators of calcium and phosphorous homeostasis. These compounds are known to stimulate, at least one of, intestinal absorption of calcium and phosphate, mobilization of bone mineral, and retention of calcium in the kidneys. Furthermore, the discovery of the presence of specific vitamin D receptors iii more than 30 tissues has led to the identification of vitamin D3 as a pluripotent regulator outside its classical role in calcium/bone homeostasis. A paracrine role for 1-alpha,25(OH)2D3 has been suggested by the combined presence of enzymes capable of oxidizing vitamin D3 into its active forms, e.g., 25-(OH)D-1α-hydroxylase, and specific receptors in several tissues such as bone, keratinocytes, placenta, and immune cells. Moreover, vitamin D3 hormone and active metabolites have been found to be capable of regulating cell proliferation and differentiation of both normal and malignant cells (Reichel, H. et al. (1989) Aim. Rev. Med. 40:71-78).

Given the activities of vitamin D3 and its metabolites, much attention has focused on the development of synthetic analogues of these compounds. A large number of these analogues involve structural modifications in the A ring, B ring, C/D rings, and, primarily, the side chain (Bouillon, R. et al. (1995) Endocr. Rev. 16(2):200-257). Although a vast majority of the vitamin D3 analogues developed to date involve structural modifications in the side chain, a few studies have reported the biological profile of A-ring diastereomers (Norman, A. W. et al. (1993) J. Biol. Chem. 268 (27):20022-20030). Furthermore, biological esterification of steroids has been studied (Hochberg, R. B. (1998) Endocr. Rev. 19(3): 331-348), and esters of vitamin D3 are known (WO 97/11053).

Moreover, despite much effort in developing synthetic analogues, clinical applications of vitamin D and its structural analogues have been limited by the undesired side effects elicited by these compounds after administration to a subject for known indications/applications of vitamin D compounds.

The activated form of vitamin D, vitamin D3, and some of its analogues have been described as potent regulators of cell growth and differentiation. It has previously been found that vitamin D3, as well as an analogue (analogue V, referred to elsewhere herein as Compound B), inhibited Benign Prostatic Hyperplasia (BPH) cell proliferation and counteracted the mitogenic activity of potent growth factors for BPH cells, such as keratinocyte growth factor (KGF) and insulin-like growth factor (IGF1). Moreover, the analogue induced bcl-2 protein expression, intracellular calcium mobilization, and apoptosis in both unstimulated and KGF-stimulated BPH cells.

U.S. Pat. No. 5,939,408 and EP808833 disclose a number of 1,25(OH)2D3 analogues including the compound 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (Compound A). U.S. Pat. No. 5,939,408 and EP808833 disclose that the compounds induce differentiation and inhibition of proliferation in various skin and cancer cell lines and are useful for the treatment of hyperproliferative skin diseases such as psoriasis, neoplastic diseases such a leukemia, breast cancer and sebaceous gland diseases such as acne and seborrheic dermatitis and osteoporosis.

The present invention is further described below with reference to the following non-limiting examples and with reference to the following figures, in which:

FIG. 1 shows synergistic or antagonistic effects on bladder cancer cell proliferation by chemotherapeutic agents in combination with calcitriol.

FIG. 2 shows combination index values, for combined treatments with calcitriol and chemotherapeutic agents in the in vitro inhibition of human bladder cancer cell line proliferation.

FIG. 3 shows VDR expression and up-regulation of CYP24 in human bladder cancer cell line incubated with calcitriol.

FIG. 4 shows inhibition of bladder cancer cell line proliferation by calcitriol.

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